Project Summary Candida albicans is a frequent commensal of the microbiota and an important opportunistic pathogen, responsible for both mucosal and systemic infections. This species is chameleon-like in its ability to adapt to different niches and to grow in different morphological forms. Here, we address the mechanism by which C. albicans undergoes phenotypic switching between ?white? and ?opaque? states. These two cell states exhibit differences in mating, filamentation, biofilm formation, immune interactions and virulence, making a molecular understanding of switching of importance to C. albicans biology. The white-opaque switch is regulated by a highly interconnected network of 6 or more master transcription factors (TFs) that act coordinately to define the two cell states. Our proposal seeks to address how prion-like domains (PrLDs) present in these TFs contribute to their function in regulating the white-opaque transcription network. We will test the hypothesis that PrLDs enable phase separation of the master TFs, thereby promoting the assembly of transcriptional complexes within liquid droplets. Researchers have proposed that a similar phase separation model underlies the transcriptional regulation of ?super-enhancers? in mammalian cells. This project therefore has the potential to determine the fundamental role of phase separation in cell fate decisions in eukaryotic cells. We provide exciting preliminary data that establishes that multiple TFs in the white-opaque circuit contain PrLDs, that recombinant forms of these TFs readily undergo efficient phase separation in vitro, and that deletion or mutation of PrLDs abrogates their function. In addition, we show that TF-TF interactions can be detected using a bimolecular fluorescence complementation (BiFC) assay that we have adapted for use in C. albicans to define protein-protein interactions. The Aims of the proposal are: AIM 1. To determine the propensity for white-opaque TFs to undergo phase separation in vitro. The 6 master TFs at the core of the white-opaque circuit will be purified and their ability to phase separate and form liquid droplets defined. Experiments will also address the precise contribution of PrLDs to the ability of TFs to undergo liquid-liquid demixing in vitro. AIM 2. To define the contribution of PrLDs to TF function in C. albicans white-opaque switching. We will determine the functional role of PrLDs by deletion of these domains and analysis of TF function in white-opaque switching assays. In addition, we will examine whether PrLDs promote protein-protein interactions by the use of BiFC assays to monitor these interactions in live C. albicans cells. Together, the proposed experiments will define the mechanism by which TFs regulate the white- opaque switch in C. albicans, and will test the exciting hypothesis that PrLDs make a key contribution to eukaryotic transcription networks by promoting phase separation and enabling TF complex formation.